Effect of Co-translational signal sequence on human SH3 display

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dc.contributor Helsingin yliopisto, Bio- ja ympäristötieteellinen tiedekunta, Bio- ja ympäristötieteellinen tiedekunta fi
dc.contributor University of Helsinki, Faculty of Biological and Environmental Sciences, Faculty of Biological and Environmental Sciences en
dc.contributor Helsingfors universitet, Bio- och miljövetenskapliga fakulteten, Bio- och miljövetenskapliga fakulteten sv
dc.contributor.author Shrestha, Subhash
dc.date.issued 2019
dc.identifier.uri URN:NBN:fi:hulib-201908283364
dc.identifier.uri http://hdl.handle.net/10138/305032
dc.description.abstract SH3 domains are relatively short and most common of modular protein binding domain in eukaryotes. They are present in proteins that play critical role in various cell signaling and regulatory pathway. Human genome encoded 296 types of SH3 domains have been successfully displayed in phagemid using classical PelB signal sequence and used for finding novel binding partners. However, given its shorter length and tendency to fold rapidly it is useful to understand if signal sequence that directs SH3 translocation through Co translational pathway is much more efficient in displaying these domains than the one that translocate protein post translationally. For the study, PelB signal sequence of phagemid displayed human SH3 library was replaced with DsbA signal sequence using round the horn PCR method (Site directed mutagenesis) and verified with agarose gel electrophoresis. Subsequently, infective phages were prepared. The infective titer of newly generated DsbAss based library was found to be higher than that of PelBss based library. Both libraries normalized at 1 x1012cfu/ml were panned against known protein targets MC159(Molluscum contagiosum 159), NCF2(Neutrophil cytosolic factor 2) and NS1(Nonstructural protein 1). Enrichment with DsbAss library was moderately higher for each antigen. However sequencing results showed that results for proteins panned with PelBss library were congruent with previous finding whereas DsbAss library selected some potential weak binders and nonspecific ones along with strong binders. Panning results of DsbAss with NCF2 was striking as all clones selected were NCF1 SH3 domains. Although further functional study was not performed. Based on the study, we concluded that both libraries have its own advantage. PelBss based library can be used for finding strong binders while DsbAss based library can be used for studying weaker interaction and functional role of NCF2-NCF1 SH3 domain interaction is still an open question. en
dc.language.iso eng
dc.publisher Helsingin yliopisto fi
dc.publisher University of Helsinki en
dc.publisher Helsingfors universitet sv
dc.subject SH3 domains en
dc.subject phage display en
dc.title Effect of Co-translational signal sequence on human SH3 display en
dc.type.ontasot pro gradu -tutkielmat fi
dc.type.ontasot master's thesis en
dc.type.ontasot pro gradu-avhandlingar sv
dc.subject.discipline perinnöllisyystiede fi
dc.subject.discipline Genetics en
dc.subject.discipline genetik sv
dct.identifier.urn URN:NBN:fi:hulib-201908283364

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